The temperature sensitive outgrowth mutants for bacterial spores will be further characterized biochemically for macromolecular synthesis, cross feeding and the effect on sequential enzyme synthesis. These mutants will be mapped to determine linkage groups. In addition, electron micrographs will be taken to define more accurately the stage of arrest. To further study the relationship between lipid and protein metabolism and outgrowth, a study will be taken of the characterization and synthesis of proteins associated with the membrane throughout the development phase. Germination of yeast spores has shown two phases of DNA synthesis; an earlier minor rise and later major rise of nuclear DNA replication. We intend to examine this by following the kinetics of nuclear, mitochondrial and nucleolar DNA synthesis during outgrowth to further understand the interrelationship between them. We have observed indirect evidence for some stable mRNA in dormant yeast spores which provides an interesting comparison to bacterial spores. The finding in our lab of the first in vivo and in vitro inhibitor of RNA polymerase provides the experimental tool needed to study the stability of mRNA in spores and during outgrowth. Several yeast protease mutants have been isolated in our laboratory. Diploids will be prepared, homozygous for these mutations, and the effect of protease deficiency on germination and outgrowth (some protein turnover occurs) will be examined.